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Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’.
In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16-18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).

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Macrophage Inflammatory Assay
巨噬细胞炎症反应分析

免疫学 > 免疫细胞功能 > 巨噬细胞
作者: Thomas J. Bartosh
Thomas J. BartoshAffiliation: Institute for Regenerative Medicine, Texas A&M University Health Science Center, Temple, USA
For correspondence: bartosh@medicine.tamhsc.edu
Bio-protocol author page: a1514
 and Joni H. Ylostalo
Joni H. YlostaloAffiliation: Institute for Regenerative Medicine, Texas A&M University Health Science Center, Temple, USA
Bio-protocol author page: a1515
Vol 4, Iss 14, 7/20/2014, 9786 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1180

[Abstract] Macrophages represent a widely distributed and functionally diverse population of innate myeloid cells involved in inflammatory response to pathogens, tissue homeostasis and tissue repair (Murray and Wynn, 2011). Macrophages can be broadly grouped into two subpopulations with opposing activites: M1 or pro-inflammatory macrophages that promote T-helper type 1 (Th1) cell immunity and tissue damage, and M2 or anti-inflammatory/alternatively activated macrophages implicated in Th2 response and resolution of inflammation. Here we describe a rapid assay we used previously to monitor changes in pro-inflammatory and anti-inflammatory cytokine production by lipopolysaccharide (LPS)-activated macrophages in response to therapeutic paracrine factors produced by adult stem cells (Bartosh et al., 2010; Ylostalo et al., 2012; Bartosh et al., 2013). The assay can be adapted appropriately to test macrophage response to other agents as well that will be referred to herein as ‘test reagents’ or ‘test compounds’.
In this protocol, the mouse macrophage cell line J774A.1 is expanded as an adherent monolayer on petri dishes allowing for the cells to be harvested easily without enzymes or cell scrapers that can damage the cells. The macropahges are then stimulated in suspension with LPS and seeded into 12-well cell culture plates containing the test reagents. After 16-18 h, the medium conditioned by the macrophages is harvested and the cytokine profile in the medium determined with enzyme-linked immunosorbent assays (ELISA). We routinely measure levels of the pro-inflammtory cytokine TNF-alpha and the anti-inflammatory cytokine interleukin-10 (IL-10).
Keywords: Macrophage(巨噬细胞), LPS(LPS), Inflammation(炎症), Prostaglandin E2(前列腺素E2), IL10(IL10)

[Abstract]

Materials and Reagents

  1. J774A.1 mouse macrophages (ATCC, catalog number: TIB-67 )
  2. 0.1 mg/ml lipopolysaccharide (LPS) (Sigma-Aldrich, catalog number: L4130 ) solution in PBS
  3. Mouse TNF-alpha Quantikine ELISA kit (R&D Systems, catalog number: MTA00B )
  4. Mouse Interleukin 10 (IL-10) Quantikine ELISA kit (R&D Systems, catalog number: M1000B )
  5. High glucose Dulbecco’s modified Eagle medium (DMEM) containing Glutamax (Life Technologies, catalog number: 10569 )
  6. Fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
  7. Penicillin-streptomycin (Life Technologies, catalog number: 15140 )
  8. Macrophage medium (see Recipes)

Equipment

  1. 150 x 15 mm petri dish (BD Biosciences, Falcon®, catalog number: 351058 )
  2. Stericup-GP 0.22 µm vacuum filtration device (EMD Millipore, catalog number: SCGPU05RE )
  3. 12-well tissue culture treated plates (Corning, catalog number: 3512 )
  4. 10 ml capacity serological pipette (VWR International, catalog number: 89130 )
  5. 1.5 ml microcentrifuge tubes
  6. 50 ml sterile conical tube (BD Biosciences, Falcon®, catalog number: 352070 )
  7. Water bath set to 37 °C
  8. Pipette-aid
  9. Centrifuge with swinging-bucket rotor and adaptors for 50-ml conical tubes
  10. Humidified cell culture incubator set to 37 °C and 5% CO2
  11. Upright microscope with 10x objective
  12. Microplate reader (capable of measuring absorbance at 450 nm, with a background correction wavelength of 540 nm or 570 nm)

Procedure

  1. Preparation of macrophage cultures
    1. Expand the J774A.1 mouse macrophages in macrophage medium on a 15 cm petri dish.
      Notes:
      1. A detailed method for culture of mouse macrophages can be found in references below.
      2. Macrophage medium should be changed every 2-3 days.
      3. The use of petri dishes allows the macrophages to be harvested for the assay without dissociating enzymes and without the requirement of a cell scraper that can be damaging to the cells.
    2. Upon reaching 70-80% confluence, aspirate the culture medium from the dish and add 10 ml of fresh macrophage medium.
    3. Harvest the macrophages by washing them from the petri dish with the macrophage medium using a pipette-aid and 10 ml serological pipette.
      Note: Spray the medium over the macrophages multiple times to dislodge the cells from the dish.
    4. Transfer the macrophage suspension from the plate to a 50 ml conical tube. Add an additional 10 ml macrophage medium to the plate and repeat wash step.
    5. Pellet the cells by centrifugation at 200-250 x g for 5-7 min.
    6. Suspend the macrophages in macrophage medium and count the cells.
      Notes:
      1.  A 15 cm petri dish containing macrophages at 70% confluence should yield approximately 15 million macrophages.
      2. The macrophages will consistently have high viability when cultured and harvested as described. Therefore, the use of a viability dye such as trypan blue for distinguishing live versus dead cells is not mandatory.
    7. Prepare a suspension of the macrophages in macrophage medium at a density of 200,000 cells per ml.

  2. Preparation of assay plates (Figure 1)
    1. Determine the number of 12-well cell culture plates necessary to assay samples of interest in triplicate.
    2. Transfer the test compound(s) of interest in triplicate to the appropriate wells of a 12-well plate.
      Note: We have routinely tested the effects of mesenchymal stem cell conditioned medium, prostaglandins such as PGE2, various pharmacological compounds, neutralizing antibodies and recombinant proteins.
    3. Add macrophage medium to all wells containing a test reagent to a final volume of 500 µl.
      Note: If the volume of the test compound employed is minute, it is advantageous to first add the macrophage medium to the wells and then transfer the test compound of interest (from step B9) to the appropriate wells. The final volume in each well should remain 500 µl.
    4. Add 500 µl macrophage medium to 6 additional wells not containing a test reagent.
      Note: These wells will provide controls for the assay.
    5. Transfer 500 µl of the macrophage cell suspension (100,000 cells) to each of 3 wells containing 500 µl of macrophage medium only (without a test reagent).
      Note: These wells will serve as unstimulated macrophage controls (Mac controls, Figure 1).


      Figure 1. Sample plate layout for the macrophage assay. To prepare the 12-well assay plate, the test reagents of interest are first added to the appropriate wells in triplicate (shown here in columns 3 and 4). Macrophage (Mac) medium is then added to every well to a final volume of 500 μl followed by addition of 500 μl of the Mac cell suspension to each well in the first column (unstimulated Mac control). 500 μl of the LPS-stimulated macrophages (LPS-Mac) are transferred to each of the remaining 9 wells (columns 2, 3, and 4). Note that three of these wells do not contain a test reagent (shown here in column 2) and therefore serve as the LPS-stimulated macrophage control (LPS-Mac control). The final volume per well is 1.0 ml. For simplicity, wells containing a vehicle control for the test reagents have been omitted. 

  3. Macrophage stimulation with LPS
    1. Stimulate the macrophages by adding a 1:500 dilution of the 0.1 mg/ml solution of LPS to the remainder of the macrophage suspension. To distribute the LPS, immediately mix the cell suspension by pipetting up and down 10-15 times.
    2. Place the cap on the 50 ml tube containing the LPS-stimulated macrophages and incubate the cells in the 50 ml tube for 5 min at room temperature.
    3. Mix the cells again then transfer 500 µl (100,000 cells) to the appropriate wells containing the test samples. Also, add 500 µl of the macrophage suspension to the three remaining wells containing 500 µl macrophage medium only (LPS-stimulated macrophage controls) (Figure 1).
    4. Rock the plate 3 times to distribute the macrophages evenly across the well and incubate at 37 °C for up to 24 h. Each well for the assay finally contains 100,000 macrophages in 1.0 ml macrophage medium and 100 ng/ml LPS (except the three unstimulated control wells).
      Note: We routinely incubate the macrophages with LPS for approximately 16-18 h. However, an abundance of macrophage-derived cytokines can be detected in the conditioned medium as early as 4-6 h after LPS stimulation.

  4. Collection of conditioned medium for assays of cytokine production
    1. Prior to collecting the medium conditioned by the macrophages, observe the cultures to verify the cells were properly activated (Figure 2).
      Note: J774A.1 macrophages stimulated with LPS appear larger, flatter, and more granular than un-stimulated macrophages.


      Figure 2. Changes in macrophage morphology in response to LPS. Brightfield images of unsimlulated macrophages (left) and macrophages stimulated with LPS for 18 h (right). Scale bar = 50 µm

    2. Collect the medium conditioned by the macrophages and place it into 1.5 ml microcentrifuge tubes.
    3. Centrifuge the samples for 5 min at 500 x g, room temperature.
    4. Transfer the supernatant to new 1.5 ml tubes.
      Notes:
      1. Although a cell pellet should not be visible, avoid touching the bottom of the tube when removing the supernatant.
      2. The samples can be used immediately for ELISA assay or aliquoted and stored at -80 °C.
    5. Vortex the medium samples prior to their use in TNF-alpha and IL-10 ELISA assays. Follow instructions provided by the manufacturer of the ELISA kits. The absorbance of the wells can be read on a microplate reader with a wavelength of 450 nm and background wavelength correction set at 540 nm or 570 nm. Representative amounts of TNF-alpha and IL-10 produced by macrophages in response to LPS and a test compound (PGE2) are shown in the graphs below (Figure 3).


      Figure 3. Effects of LPS and PGE2 on TNF-alpha and IL-10 production by macrophages. Macrophages were stimulated for 18 h with 100 ng/ml LPS alone or LPS in combination with 1 ng/ml of the immune modulatory factor prostaglandin E2 (PGE2). LPS increases levels of the pro-inflammatory cytokine TNF-alpha produced by the macrophages. PGE2 attenuates macrophage production of TNF-alpha elicited by LPS stimulation but enhances levels of the anti-inflammatory cytokine IL-10. Values are mean +/- SD, n=3 per group.

Notes

  1. We have routinely used mouse TNF-alpha and IL-10 ELISA kits to monitor the specific activity of mouse macrophages in response to paracrine factors, such as PGE2, produced by mesenchymal stem cells. However, production of other macrophage-derived cytokines is also increased by LPS stimulation including CXCL2/MIP2, MCP-1, IL-6, IL-1 beta, and IL12-p40 (Ylostalo et al., 2012). These cytokines can also be measured by ELISA.
  2. When the assay is performed as described above, levels of TNF-alpha in the conditioned medium increases from 10-30 pg/ml in unstimulated macrophage cultures to 800-1,200 pg/ml in macrophages stimulated for 16-18 h with LPS. Levels of IL-10 increase only slightly with LPS stimulation. However, IL-10 levels can be markedly enhanced in LPS-stimulated macrophage cultures by factors that promote an anti-inflammatory macrophage phenotype such as PGE2 (Figure 3).
  3. Different preparations of LPS can elicit variations in macrophage cytokine production. The concentration of LPS used in the assay can be adjusted to achieve the desired results. We have routinely used LPS concentrations ranging from 10 ng/ml to 1 µg/ml.

Recipes

  1. Macrophage medium (500 ml)
    High glucose DMEM containing Glutamax, 445 ml
    Fetal bovine serum, 50 ml
    Penicillin-streptomycin, 5 ml
    Filter sterilize (0.22 µm) and store up to one month at 4 °C
    Prewarm to 37 °C in water bath immediately prior to use

Acknowledgments

This protocol was adapted from our original work (Bartosh et al., 2013; Ylostalo et al., 2012) that was supported by grants from the NIH (P40RR17447) and Cancer Prevention & Research Institute of Texas (RP110620) to Darwin J. Prockop, Institute for Regenerative Medicine, Texas A&M University Health Science Center.

References

  1. Bartosh, T. J., Ylostalo, J. H., Bazhanov, N., Kuhlman, J. and Prockop, D. J. (2013). Dynamic compaction of human mesenchymal stem/precursor cells into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1). Stem Cells 31(11): 2443-2456.
  2. Bartosh, T. J., Ylostalo, J. H., Mohammadipoor, A., Bazhanov, N., Coble, K., Claypool, K., Lee, R. H., Choi, H. and Prockop, D. J. (2010). Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107(31): 13724-13729.
  3. Murray, P. J. and Wynn, T. A. (2011). Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11(11): 723-737.
  4. Ylostalo, J. H., Bartosh, T. J., Coble, K. and Prockop, D. J. (2012). Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype. Stem Cells 30(10): 2283-2296.

材料和试剂

  1. J774A.1小鼠巨噬细胞(ATCC,目录号:TIB-67)
  2. 0.1mg/ml溶于PBS中的脂多糖(LPS)(Sigma-Aldrich,目录号:L4130)
  3. 小鼠TNF-αQuantikine ELISA试剂盒(R& D Systems,目录号:MTA00B)
  4. 小鼠白细胞介素10(IL-10)Quantikine ELISA试剂盒(R& D Systems,目录号:M1000B)
  5. 含有Glutamax(Life Technologies,目录号:10569)的高葡萄糖Dulbecco改良的Eagle培养基(DMEM)
  6. 胎牛血清(Atlanta Biologicals,目录号:S11550)
  7. 青霉素 - 链霉素(Life Technologies,目录号:15140)
  8. 巨噬细胞培养基(见配方)

设备

  1. 150×15mm培养皿(BD Biosciences,Falcon ,目录号:351058)
  2. Stericup-GP0.22μm真空过滤装置(EMD Millipore,目录号:SCGPU05RE)
  3. 12孔组织培养处理板(Corning,目录号:3512)
  4. 10ml容量血清移液管(VWR International,目录号:89130)
  5. 1.5 ml微量离心管
  6. 50ml无菌锥形管(BD Biosciences,Falcon ,目录号:352070)
  7. 水浴设置为37℃
  8. 移液助剂
  9. 带有摆动斗式转子的离心机和适用于50 ml锥形管的适配器
  10. 将加湿的细胞培养箱设定为37℃和5%CO 2/h
  11. 带有10倍物镜的直立显微镜
  12. 酶标仪(能够测量在450nm处的吸光度,背景校正波长为540nm或570nm)

程序

  1. 巨噬细胞培养物的制备
    1. 在15cm培养皿上的巨噬细胞培养基中扩增J774A.1小鼠巨噬细胞 注意:
      1. 下面的参考文献中可以找到小鼠巨噬细胞培养的详细方法。
      2. 使用培养皿允许巨噬细胞被收获   测定而不解离酶并且不需要a 可破坏细胞的细胞刮刀。
    2. 达到70-80%汇合时,从培养皿中吸出培养基并加入10ml新鲜巨噬细胞培养基。
    3. 收获巨噬细胞通过从培养皿中洗涤他们 巨噬细胞培养基,使用移液管和10ml血清移液管 注意:将培养基多次喷在巨噬细胞上以从培养皿中取出细胞。
    4. 将巨噬细胞悬浮液从板转移到50毫升锥形 管。 添加另外10毫升巨噬细胞培养基板和重复 洗涤步骤。
    5. 通过在200-250×g离心5-7分钟来沉淀细胞
    6. 将巨噬细胞悬浮在巨噬细胞培养基中并计数细胞 注意:
      1. 一个15厘米的培养皿含有70%汇合的巨噬细胞。
      2. 巨噬细胞在培养时将始终具有高存活力 并如所述收获。 因此,使用活力染料等 因为台盼蓝用于区分活细胞和死细胞不是 强制性。
    7. 准备巨噬细胞在巨噬细胞培养基中的悬浮液,密度为200,000细胞/ml。

  2. 测定板的制备(图1)
    1. 确定一式三份测定感兴趣样品所需的12孔细胞培养板的数目
    2. 将感兴趣的测试化合物一式三份转移到12孔板的合适孔中 注意:我们已经常规测试间充质干细胞的作用 条件培养基,前列腺素如PGE2,各种药理学   化合物,中和抗体和重组蛋白。
    3. 向含有试验试剂的所有孔中加入巨噬细胞培养基至终体积为500μl 注意:   如果所用的测试化合物的体积是微小的,则是 有利的是首先将巨噬细胞培养基加入孔中 将感兴趣的测试化合物(来自步骤B9)转移到合适的   井。 每个孔中的最终体积应保持500μl。
    4. 向不含试验试剂的6个孔中加入500μl巨噬细胞培养基 注意:这些孔将提供测定的控制。
    5. 转移500微升的巨噬细胞悬浮液(100,000个细胞) 每个3孔含有500μl的巨噬细胞培养基(无a 测试试剂)。
      注意:这些孔将用作未刺激的巨噬细胞对照(Mac对照,图1)。


      图  1.巨噬细胞测定的样品板布局。准备12孔  测定板,首先将感兴趣的测试试剂加入 合适的孔一式三份(这里显示在第3和4列)。 然后将巨噬细胞(Mac)培养基加入到每个孔中至终体积  500μl,然后加入500μl的Mac细胞悬浮液 第一列中的每个孔(未刺激的Mac对照)。 500μl的 LPS刺激的巨噬细胞(LPS-Mac)转移到每个 剩余9个孔(第2,3和4列)。注意这些井中的三个 不包含测试试剂(如第2列所示),因此 作为LPS刺激的巨噬细胞对照(LPS-Mac对照)。 的 每孔的最终体积为1.0ml。 为简单起见, 试验试剂的车辆控制已被省略。

  3. 巨噬细胞用LPS刺激
    1. 通过加入0.1 mg/ml的1:500稀释液刺激巨噬细胞 LPS的溶液到巨噬细胞悬浮液的剩余部分。 至 分配LPS,立即通过吹吸混合细胞悬浮液 并下降10-15次。
    2. 将盖放在50ml管上 含有LPS刺激的巨噬细胞并孵育细胞 50ml管在室温下温育5分钟
    3. 再次混合细胞 然后转移500微升(100,000个细胞)到合适的孔中   测试样品。 另外,加入500μl的巨噬细胞悬浮液 三个剩余的孔仅含有500μl巨噬细胞培养基 (LPS刺激的巨噬细胞对照)(图1)
    4. 摇滚 板3次以将巨噬细胞均匀地分布在孔中 在37℃孵育长达24小时。 每个孔最后进行测定 在1.0ml巨噬细胞培养基和100ng/ml中含有100,000个巨噬细胞 LPS(除了三个未刺激的对照孔) 注意:我们 常规地将巨噬细胞与LPS孵育约16-18小时。 然而,可以检测到巨噬细胞衍生的细胞因子的丰度 条件培养基早在LPS刺激后4-6小时

  4. 收集用于测定细胞因子产生的条件培养基
    1. 在收集由巨噬细胞调节的培养基之前,观察 培养物以验证细胞是否被适当激活(图2) 注意:用LPS刺激的J774A.1巨噬细胞看起来比未刺激的巨噬细胞更大,更扁平,更颗粒。


      图   2.响应LPS的巨噬细胞形态的变化。 Brightfield 未刺激的巨噬细胞(左)和刺激的巨噬细胞的图像 与LPS 18小时(右)。 比例尺=50μm

    2. 收集由巨噬细胞调节的培养基,并将其置于1.5ml微量离心管中
    3. 在500×g/hr,室温下离心样品5分钟
    4. 将上清液转移到新的1.5ml试管中 注意:
      1. 虽然细胞沉淀不应该可见,但是在去除上清液时,应避免接触管底。
      2. 样品可立即用于ELISA测定,或等分并保存在-80℃下。
    5. 涡旋培养基样品在其用于TNF-α和IL-10 ELISA测定。按照制造商提供的说明 ELISA试剂盒。可以在微量培养板上读取孔的吸光度 阅读器,波长为450nm,背景波长校正 设置在540nm或570nm。代表量的TNF-α和IL-10 由巨噬细胞响应LPS和测试化合物(PGE2) 如下图所示(图3)。


      图3. LPS的作用 和PGE2对巨噬细胞TNF-α和IL-10产生的影响。 巨噬细胞 用100ng/ml单独的LPS或LPS组合刺激18小时 与1ng/ml的免疫调节因子前列腺素E2(PGE2)。 LPS增加产生的促炎细胞因子TNF-α的水平  由巨噬细胞。 PGE2减弱TNF-α的巨噬细胞产生 引起LPS刺激,但增强抗炎的水平  细胞因子IL-10。值是平均值±SD,每组n = 3。

笔记

  1. 我们常规使用小鼠TNF-α和IL-10 ELISA试剂盒来监测小鼠巨噬细胞对间充质干细胞产生的旁分泌因子(如PGE2)反应的比活性。然而,其它巨噬细胞衍生的细胞因子的产生也通过LPS刺激增加,包括CXCL2/MIP2,MCP-1,IL-6,IL-1β和IL12-p40(Ylostalo等人, 2012)。这些细胞因子也可以通过ELISA测量
  2. 当如上所述进行测定时,条件培养基中TNF-α的水平从未刺激的巨噬细胞培养物中的10-30pg/ml增加至用LPS刺激的巨噬细胞中的800-1,200pg/ml。 LPS刺激时IL-10的水平仅轻微增加。然而,通过促进抗炎巨噬细胞表型的因子如PGE2,在LPS刺激的巨噬细胞培养物中IL-10水平可以显着增强(图3)。
  3. LPS的不同制备物可引起巨噬细胞细胞因子产生的变化。可以调节在测定中使用的LPS的浓度以实现期望的结果。我们常规使用LPS浓度范围为10ng/ml至1μg/ml

食谱

  1. 巨噬细胞培养基(500ml) 高葡萄糖DMEM含Glutamax,445 ml
    胎牛血清,50ml
    青霉素 - 链霉素,5ml /孔 过滤灭菌(0.22μm),并在4℃下储存一个月。
    使用前立即在37℃水浴中预热,

致谢

该方案改编自我们来自NIH(P40RR17447)和癌症的捐赠支持的原始工作(Bartosh等人,2013; Ylostalo等人,2012年) 预防& 德克萨斯研究所 (RP110620)至Darwin J.Prockop,Institute for Regenerative Medicine,Texas A& M University Health Science Center。

参考文献

  1. Bartosh,T.J.,Ylostalo,J.H.,Bazhanov,N.,Kuhlman,J.and Prockop,D.J。(2013)。 将人间质干细胞/前体细胞动态压实成球体自激活半胱天冬酶依赖性IL1信号以增强分泌炎症和免疫调节剂(PGE2,TSG6和STC1)。 干细胞 31(11):2443-2456。
  2. Bartosh,T.J.,Ylostalo,J.H.,Mohammadipoor,A.,Bazhanov,N.,Coble,K.,Claypool,K.,Lee,R.H.,Choi,H.and Prockop,D.J。(2010)。 人间质基质细胞(MSC)聚集成3D球状体增强了它们的抗炎性质。 Proc Natl Acad Sci USA 107(31):13724-13729。
  3. Murray,P.J。和Wynn,T.A。(2011)。 巨噬细胞亚群的保护性和致病性功能。 Nat Rev Immunol 11(11):723-737。
  4. Ylostalo,J.H.,Bartosh,T.J.,Coble,K.and Prockop,D.J。(2012)。 作为球体培养的人间充质干/基质细胞是自激活的 产生前列腺素E2,其指导受刺激的巨噬细胞进入抗炎表型。干细胞 30(10):2283-2296。
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How to cite this protocol: Bartosh, T. J. and Ylostalo, J. H. (2014). Macrophage Inflammatory Assay. Bio-protocol 4(14): e1180. DOI: 10.21769/BioProtoc.1180; Full Text



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